PROJECT SUMMARY This proposal describes a five-year career development program to prepare Dr. Haodi Wu for a career as an independent investigator. This program will build on Dr. Haodi Wu?s background as a cell biologist and biophysicist by providing him expertise in molecular biology techniques such as sequencing technology and genome-editing to advance our understanding of the pathogenesis and treatment of dilated cardiomyopathy (DCM). Dr. Haodi Wu will be mentored by Dr. Joseph Wu, professor and Director of Stanford Cardiovascular Institute and a pioneer of stem cell biology research and cardiovascular disease modeling with iPSC derived cardiomyocytes (iPSC-CMs), and co-mentored by Dr. Michael Snyder, professor and Chair of Stanford Genetics Department and a world-renowned expert in omics and personalized medicine. The K99 phase of Dr. Haodi Wu?s training will consist of structured mentorship by the primary mentor and co-mentor, close interactions with collaborators, complementary meeting with advisory committee, formal coursework, a provocative research project, and a program of career transition. Patient specific iPSC-CMs present a unique opportunity to define the cellular phenotype, elucidate pathology mechanism and develop potential treatment of DCM. In Dr. Haodi Wu?s previous works, he generated iPSC-CM models from a DCM family, and showed patient specific iPSC-CMs recapitulate key phenotype and molecular cues of DCM. He also identified the subtype-specific epigenetic activation of phosphodiesterase (PDEs) as a key molecular regulation that contributes to DCM pathogenesis. Moreover, he established multiple signaling and functional assays on iPSC-CMs, which allow for examination of the molecular mechanism in DCM iPSC-CM models with a level of depth and resolution never before achieved. With the current advancement in high-throughput sequencing and the cutting-edge CRISPR/dCas9 molecular tools, Dr. Haodi Wu is in a unique position to uncover the unknown mechanisms that underlie the remodeled PDE expression pattern in human DCM cardiomyocytes, and to develop novel molecular approaches and compounds that target molecular basis of DCM in a gene and regulatory element specific way. In the K99 phase, Dr. Haodi Wu will generate and characterize genome edited DCM iPSC-CM models via introduction of dominant negative mutations (Aim1). With the platform, Dr. Haodi Wu will integrate both RNA-seq and ChIP-seq data to identify the key epigenetic regulatory mechanism of PDE expression in DCM cardiomyocytes with TNNT2 mutation (Aim2). Basing on the knowledge, he will develop novel molecular approaches and small molecules that restore PDE expression and signaling in R00 phase (Aim3). Collectively, Dr. Haodi Wu?s proposed work will create a valuable platform to study the detailed role of single DCM mutation in human cardiomyocytes, and reveal mutation-specific molecular mechanism of PDE regulations underlying DCM pathogenesis. Additionally, this work will cast new light on the application of novel molecular tools and PDE inhibitors in targeting on the molecular basis of DCM disease, which will be carried out by Dr. Haodi Wu as an independent investigator.